9-cis retinoic acid accelerates calcitriol-induced osteocalcin production and promotes degradation of both vitamin D receptor and retinoid X receptor in human osteoblastic cells.

Abstract vitamin D receptor (VDR) and retinoid X receptor (RXR) heterodimerize to mediate the genomic actions of 1alpha,25-dihydroxyvitamin D(3) (1alpha,25(OH)(2)D(3), calcitriol), the biologically active form of vitamin D(3). In this study, we show that 9-cis retinoic acid (9-cisRA), the ligand for RXR, accelerates calcitriol-induced expression of osteocalcin gene, the marker for mature osteoblasts. Calcitriol and its synthetic analog KH1060 (1 nM) induced osteocalcin secretion after a 96-h incubation period as detected by radioimmunoassay. When these compounds were used together with 9-cisRA, osteocalcin protein secretion was, however, detected already after 72 and 48 h, respectively. Detection of osteocalcin mRNA with quantitative PCR revealed elevated mRNA levels already after a 4-h treatment of the cells with calcitriol, KH1060, or 9-cisRA compared with untreated cells. In combination treatments, 9-cisRA rapidly stimulated osteocalcin mRNA synthesis induced by the different vitamin D(3) compounds. In MG-63 cells treated with calcitriol or KH1060, the stimulation was maximal after the first 4 h and diminished thereafter. In fact, after the 48-h incubation 9-cisRA reduced osteocalcin mRNA levels in KH1060-treated cells, the amount of mRNA being only 44% of the levels obtained with KH1060 alone. The reduction was accompanied by an increased degradation rate of both VDR and RXRbeta in the presence of 9-cisRA. Furthermore, 9-cisRA increased the formation of RXRbeta-VDR-VDRE complex on the osteocalcin gene VDRE. These results suggest that 9-cisRA accelerates calcitriol-induced osteocalcin production in human osteoblastic cells through increased formation of transcriptionally active chromatin complexes and, subsequently, promotes degradation of the heterodimeric complex of VDR and RXR.